Phospholipids consist of a glycerol backbone with two esterified fatty acids in an outer (sn-1) and the middle (sn-2) position, while the third hydroxyl group of the glycerol is esterified with phosphoric acid. The phosphoric acid may, in turn, be esterified to for example an amino alcohol like ethanolamine(phosphatidylethanolamine), choline (phosphatidylcholine). The third hydroxyl group may also, instead of being esterified with phosphoric acid, be bound to sugar residues such a galactose or a dimer thereof such as in digalactosyldiglyceride.
Phospholipases are defined herein as enzymes that participate in the hydrolysis of one or more bonds in the phospholipids including digalactosyldiglyceride described above.
Several types of phospholipase activity can be distinguished which hydrolyse the ester bond(s) that link the fatty acyl moieties to the glycerol backbone.                Phospholipase A1 (EC 3.1.1.32) and A2 (EC 3.1.1.4) catalyse the deacylation of one fatty acyl group in the sn-1 and sn-2 positions respectively, from a diacylglycerophospholipid to produce a lysophospholipid.        Lysophbspholipase (EC 3.1.1.5—also called phospholipase B by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (Enzyme Nomenclature, Academic Press, New York, 1992)) catalyses the hydrolysis of the remaining fatting acyl group in a lysophospholipid. A phospholipase B has been reported from Penicillium notatum (Saito et al., 1991, Methods in Enzymology 197:446-456), which catalyses the deacylation of both fatty acids from a diacylglycerophospholipid and intrinsically possesses lysophospholipase activity.        Galactolipase (EC 3.1.4.3) catalyses the hydrolysis of one or both fatty acyl group in the sn-1 and sn-2 positions respectively, from a digalactosyldiglyceride.Phospholipase C (EC 3.1.4.3) hydrolyses the phosphate ester bond between the glycerol backbone and the phosphate group, for example:phosphatidylcholine+H2O=1,2 diacylglycerol+choline phosphate.Phospholipase D (EC 3.1.4.4) hydrolyses the phosphate ester bond between the phosphate group and the amine alcohol, for example: phosphatidylcholine+H2O choline+phosphatidic acid.        
Phospholipases may conveniently be produced in microorganisms. Microbial phospholipases are available from a variety of sources; Bacillus species are a common source of bacterial enzymes, whereas fungal enzymes are commonly produced in Aspergillus species.
Fungal enzymes with phospholipase activity have been reported from various sources, including Cryptococcus neoformans (Chen et al., 1997, Infection and Immunity 65:405-411), Fusobacterium necrophorum (Fifis et al., 1996, Veterinary Microbiology 49:219-233), Penicillium notatum (also known as Penicillium chrysogenum; Kawasaki, 1975, Journal of Biochemistry 77:1233-1244; Masuda et al., 1991, European Journal of Biochemistry 202:783-787), Penicillium cyclopium (Mustranta et al., 1995, Process Biochemistry 30:393-401), Saccharomyces cerevisiae (Ichimasa et al., 1985, Agric. Biol. Chem. 49:1083-1089; Paultauf et al., 1994, J. Biol. Chem. 269:19725-19730), Torulaspora delbrueckii (old name Saccharomyces rosei, Kuwabara, 1988, Agric. Biol. Chem. 52:2451-2458; Watanabe et al., 1994, REMS Microbiological Letters 124:29-34), Neurospora crassa (Chakravarti et al., 1981, Archives of Biochemistry and Biophysics 206:393-402), Aspergillus niger (Technical Bulletin, G-zyme™ G6999, Enzyme Bio-Systems Ltd.; Mustranta et al., 1995, supra), Corticium centrifugum (Uehara et al., 1979, Agric. Biol. Chem. 43:517-525), Fusarium oxysporum (WO 98/26057), and Fusarium solani (Tsung-Che et al., 1968, Phytopathological Notes 58:1437-38).
Fungal phospholipase genes have been cloned from several sources including Penicillum notatum (Masuda et al., 1991, supra), Torulaspora delbrueckii (Watanabe et al., 1994, FEMS Microbiology Letters 124: 29-34), Saccharomyces cerevisiae (Lee at al., 1994, Journal of Biological Chemistry 269: 19725-19730), Aspergillus (JP 10155493), Neurospora crassa (EMBL 042791), and Schizosaccharomyces pombe (EMBL 013857).
Phospholipases may be used in a manifold of industrial applications, including for the modification of phospholipid emulsifiers. An example of a phospholipid emulsifier is lecithin, which is a mixture of both polar and neutral lipids in which the content of polar lipids is at least 60%. Phospholipid emulsifiers have many food and non-food applications, for example egg-lecithin is used as an emulsifier in for example dairy products, specifically mayonnaise, dressings, pastry, etc., soya lecithin for example, is for example used as an emulsifier in (low calorie) sauces, bread, margarine, cosmetics etc, other lecithins are used in for example chocolates, calf feed. Modification of phospholipid emulsifiers by phospholipases may cause an increased emulsification of the oil/water mixture. Modification of phospholipid emulsifiers by phospholipases may increase the stability of the emulsions resulting from the addition of the modified phospholipid emulsifiers for a wider or different pH and/or temperature range. Modification of phospholipid emulsifiers by phospholipases may increase the stability of the emulsions, resulting from the addition of modified phospholipid emulsifiers, in the presence of Ca2+ or Mg2+ ions.
Another example of industrial application of phospholipases is that they can be used for the degumming of vegetable oils in the processing of these oils. In a typical degumming process, lecithins are removed from vegetable oils, for example soy oils, rapeseed (canola) oils, linseed oils, sunflower oils, to increase among others the stability of the vegetable oil, by washing the oil phase with water, wherein mixing of the water and oil under high shear conditions forces the bulk of the lecithins into the aqueous phase, which is subsequently removed in a separator. In this so-called water degumming phase, only the rapidly hydratable phospholipids are readily removed, for example phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. The non-hydratable phopholipids/phosphatides, mostly the phospholipids, which consist of up to 50% of magnesium and/or calcium salts cannot readily be removed in the water degumming step. Exposure of the non-hydratable phopholipids/phosphatides to Phospholipase A2 makes these phospholipids more soluble in water and therefore easier to extract in a water degumming phase. Another example of industrial application of phospholipases is that they are used to remove the precipitate that occurs during the saccharification (with the aid of α-amylase and glucoamylase) of wheat gluten or wheat starch to produce glucose syrups. The removal of the precipitate considerably speeds up the subsequent filtration of the resulting glucose syrups. The above-mentioned industrial applications of the phospholipase enzyme are only a few examples and this listing is not meant to be restrictive.
Yet another example of an industrial application of phospholipases in food is that phospholipases are particularly useful in baking applications to improve dough or baked product quality. Wheat flour contains approximately 2.2-2.9% lipids. The flour lipids can be divided into starch lipids (0.8-0.9%) and non-starch lipids (1.4-2.0%). Whereas the starch lipids consist mainly of polar lysophospholipids, the non-starch lipids consist of about 40% neutral triglycerides and 40% polar phospho- and glycolipids. For optimisation of the flour lipids fraction it is possible to hydrolyse the phospholipids in situ in the dough by adding phospholipase A.
For example EP-A-109244 and WO98/26057 describe this use of phospholipase A in breadmaking. In Czechoslovakian patent AO 190 264 phosphatidic acid (product of phospholipase D hydrolysis) is applied as dough and bread improving agent. In EP-A-075463 the combination of phospholipase A and phospholipase D is applied to produce lysophosphatidic acid as a dough-conditioning agent.
WO 00/32758 describes the production of lipolytic enzyme variants by making alterations to the amino acid sequence of the lipolytic enzyme so as to increase the level of desired activity. For baking applications the variant from the lipolytic enzyme of Humicula family or the Zygomycetes family was found to be particularly useful because it appeared to have phospholipase and/or digalactosyldiglyceride activity. WO 98/45453 describes a polypeptide having lipase activity derivable from Aspergillus tubigensis which is also showing high hydrolytic activity on digalactosyldiglyceride. These enzymes, however, suffer from a relatively low specific activity.
In the above processes, it is advantageous to use phospholipases that are obtained by recombinant DNA techniques. Such recombinant enzymes have a number of advantages over their traditionally purified counterparts. Recombinant enzymes may be produced at a low cost price, high yield, free from contaminating agents like bacteria or viruses but also free from bacterial toxins or contaminating other enzyme activities.
The present invention addresses at least one if not all of the above problems.